All cells require oxygen for proper functioning. A coordinated transcriptional response to decreased oxygen levels (hypoxia) maintains homeostasis through short-term and long-term adaptive mechanisms. Two structurally similar transcription factors, Hypoxia-Inducible Factor 1 and 2 (HIF-1 and HIF-2), are thought to be the primary mediators of hypoxia-induced gene expression. The HIFs play critical roles in normal development, control of red blood cell production; wound healing, and response to ischemic diseases. Unfortunately, they also play important roles in promoting tumor growth. Mounting evidence suggests that HIF-1 and HIF-2 are not identical in function, although the mechanistic basis for these differences is not known. Using an emerging technology, ChIP-chip, we have comprehensively identified all regions of the genome that are bound by HIF-1, and have integrated these data with gene expression profiling to establish a functional relationship between HIF-1 binding and gene transactivation. In the first Specific Aim of this proposal, we will use the same approaches to identify HIF-2 binding sites and gene transactivation. We will determine the effects of specific loss of HIF-1 and HIF-2 on gene expression, and will search for mechanisms that dictate differences between HIF-1 and HIF-2 in their DNA binding and gene transactivation. Integrating these diverse datasets, we will establish a comprehensive view of how HIF-1 and HIF-2 are similar and how they differ in their primary function as transcriptional activators. Gene expression is also greatly influenced by the localized structure of chromatin. In the second Specific Aim, we will use ChIP-chip to map the baseline and hypoxia-induced changes in specific histone modifications, including acetylation and methylation, which regulate chromatin structure. When integrated with HIF-1 binding, HIF-2 binding, and gene expression profiles, these global and gene-specific studies will establish an understanding of the network of trans-acting and epigenetic mechanisms regulating gene expression in response to hypoxia. In particular, we wish to define how epigenetics modulate HIF binding to DNA, how epigenetics impact gene expression by HIF, and in turn how hypoxia/HIF alters epigenetics. This knowledge is critical as strategies to increase or decrease hypoxic gene expression progress into clinical testing.